S produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.[Keywords: Adhesion; c-di-GMP; flagella; GGDEF; EAL; RpoS] Supplemental material is available at http://www.genesdev.org.

SummaryBis-(3Ј-5Ј)-cyclic-di-guanosine monophosphate (c-di-GMP) is a bacterial signalling molecule produced by diguanylate cyclases (DGC, carrying GGDEF domains) and degraded by specific phosphodiesterases (PDE, carrying EAL domains). Neither its full physiological impact nor its effector mechanisms are currently understood. Also, the existence of multiple GGDEF/EAL genes in the genomes of most species raises questions about output specificity and robustness of c-di-GMP signalling. Using microarray and gene fusion analyses, we demonstrate that at least five of the 29 GGDEF/EAL genes in Escherichia coli are not only stationary phase-induced under the control of the general stress response master regulator s S (RpoS), but also exhibit differential control by additional environmental and temporal signals. Two of the corresponding proteins, YdaM (GGDEF only) and YciR (GGDEF + EAL), which in vitro show DGC and PDE activity, respectively, play an antagonistic role in the expression of the biofilm-associated curli fimbriae. This control occurs at the level of transcription of the curli and cellulose regulator CsgD. Moreover, we show that H-NS positively affects curli expression by inversely controlling the expression of ydaM and yciR. Furthermore, we demonstrate a temporally fine-tuned GGDEF cascade in which YdaM controls the expression of another GGDEF protein, YaiC. By genome-wide microarray analysis, evidence is provided that YdaM and YciR strongly and nearly exclusively control CsgD-regulated genes. We conclude that specific GGDEF/EAL proteins have very distinct expression patterns, and when present in physiological amounts, can act in a highly precise, non-global and perhaps microcompartmented manner on a few or even a single specific target(s).

Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The operon is present in many bacteria, including species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogenactivated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Switching from the motile planktonic bacterial lifestyle to a biofilm existence is stimulated by the signalling molecule bis-(3′-5′)-cyclic-diguanosine monophosphate (cyclic-di-GMP), which is antagonistically controlled by diguanylate cyclases (DGCs; characterized by GGDEF domains) and specific phosphodiesterases (PDEs; mostly featuring EAL domains). Here, we present the expression patterns of all 28 genes that encode GGDEF/EAL domain proteins in Escherichia coli K-12. Twenty-one genes are expressed in Luria–Bertani medium, with 15 being under σ S control. While a small subset of GGDEF/EAL proteins (YeaJ and YhjH) is dominant and modulates motility in post-exponentially growing cells, a diverse battery of GGDEF/EAL proteins is deployed during entry into stationary phase, especially in cells grown at reduced temperature (28 °C). This suggests that multiple signal input into cyclic-di-GMP control is particularly important in growth-restricted cells in an extra-host environment. Six GGDEF/EAL genes differentially control the expression of adhesive curli fimbriae. Besides the previously described ydaM, yciR, yegE and yhjH genes, these are yhdA (csrD), which stimulates the expression of the DGC YdaM and the major curli regulator CsgD, and yeaP, which contributes to expression of the curli structural operon csgBAC. Finally, we discuss why other GGDEF/EAL domain-encoding genes, despite being expressed, do not influence motility and/or curli formation.

RprA is a small regulatory RNA known to weakly affect the translation of σS (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase-induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5′-untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base-pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD-activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off – for instance, later in stationary phase or during biofilm formation – or by conditions that further activate RprA expression via the Rcs two-component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.

Upon environmental changes, bacteria reschedule gene expression by directing alternative sigma factors to core RNA polymerase (RNAP). This sigma factor switch is achieved by regulating relative amounts of alternative sigmas and by decreasing the competitiveness of the dominant housekeeping r 70 . Here we report that during stationary phase, the unorthodox Crl regulator supports a specific sigma factor, r S (RpoS), in its competition with r 70 for core RNAP by increasing the formation of r S -containing RNAP holoenzyme, Er S . Consistently, Crl has a global regulatory effect in stationary phase gene expression exclusively through r S , that is, on r S -dependent genes only. Not a specific promoter motif, but r S availability determines the ability of Crl to exert its function, rendering it of major importance at low r S levels. By promoting the formation of Er S , Crl also affects partitioning of r S between RNAP core and the proteolytic r S -targeting factor RssB, thereby playing a dual role in fine-tuning r S proteolysis. In conclusion, Crl has a key role in reorganising the Escherichia coli transcriptional machinery and global gene expression during entry into stationary phase.

There is burgeoning interest in protein microarrays, but a source of thousands of nonredundant, purified proteins was not previously available. Here we show a glass chip containing 2413 nonredundant purified human fusion proteins on a polymer surface, where densities up to 1600 proteins/cm 2 on a microscope slide can be realized. In addition, the polymer coating of the glass slide enables screening of protein interactions under nondenaturing conditions. Such screenings require only 200-l sample volumes, illustrating their potential for high-throughput applications. Here we demonstrate two applications: the characterization of antibody binding, specificity, and cross-reactivity; and profiling the antibody repertoire in body fluids, such as serum from patients with autoimmune diseases. For the first application, we have incubated these protein chips with anti-RGSHis 6 , anti-GAPDH, and anti-HSP90␤ antibodies. In an initial proof of principle study for the second application, we have screened serum from alopecia and arthritis patients. With analysis of large sample numbers, identification of disease-associated proteins to generate novel diagnostic markers may be possible.